3beta,5,6 - trihydroxy - 6 - hydroxymethyl-pregnan-20-one,derivatives thereof and intermediates thereto



Patented Apr. 28, 1970 A specific example is the reaction of6,8-acetoxymethyl- 3,509,137 3{3-benzyloxy-5a,6a-epoxypregnan 20 one20-ethylene 35,5,6 TRIHYDROXY 6 HYDROXYMETHYL- ketal, obtained byacetylation of the corresponding 6fi- THEREOF hydroxymethyl compound,with perchloric acid in aque- Leonir d filgsted, Highland Park, 111.,assignor to G. D. 5 Gus acetoneflthus Producing mixture 9 i ep 1mg:Searle & Co., Chicago, 111., a corporation of D l 5,6-diols, which areseparated by crystallization to yield No Drawing. Filed Oct. 17, 1968,Ser. No. 768,528 r acetoxymethyl B benzyloxy 5 m y y- Int. Cl. C07c169/32, 173/00 pregnan-ZO-one and 6u-acetoxymethyl-3fl-benzyloxy 5oz,

U.S. Cl. 260-23955 10 Claims 6p-dihydroxypregnan-ZO-one.

10 The instant 3,8-hydroxy compounds are readily obtained by removal ofthe benzyl group of the aforementioned 3i3-benzyloxy substances.Catalytic hydrogenolysis ABSTRACT OF THE DISCLOSURE Th ti f 5 h d -6- idith 11. provides a particularly convenient method and palladiummethylsulfoxonium methylide affords the corresponding 13 a PE Catalyst ly 5' y P 3f' 5a,6u-epoxy 6B hydroxymethyl derivatives which are 15 fi' YYPE E 111 aqueous acetlc aclfl 15 cleaved by means of a suitable acid oralkaline reagent to thus Shaken Wlth 5% palladlum-onjcarbon Catalyst Ina yield the 5,6-dihydroxy-6-hydroxymethyl compounds. The hydrogenatmosphere at atmosphenc Pressure and I001?! latter substances displayuseful pharmacological propertemperature to afford 6a-acet0xymethy1 5 5-ties, e.g. pepsin-inhibitory and anti-protozoal. Y YP The 3-ketocompounds of this invention are conveniently produced by reaction of thecorresponding 35- The present invention is concerned with novel chem- YY Substances With a Suitable OXidiZiHg agent ical compounds belonging tothe pregnane family of y Y fi Y YP n lIl Steroids and characterized by a5 h acetone is thus contacted with aqueous chromic acid tomethyl/acyloxymethyl structure and with the 50,6u- Yleld P Y- 1 Y YP P-Q epoxy 6B hydroxymethyl/acyloxymethyl precursors The 5,6'd1hYdY0XYcompfmnds 0f th 1S ufl n d18- thereof. Those 5,6-diols are representedby the following P Y p l fi P P f lllllstrattfi structured f ula bytheir ability to inhibit the enzymatic action of pepsin CH and alsotheir ability to inhibit the growth of microorganisms such as theprotozoan Tezrahymena gellez'i. a The pepsin-inhibitory property of theinstant com- CH pounds is specifically illustrated by the activity of60:-

acetoxymethyl 3 fi,5a,6/3 trihydroxypregnan-ZO-one and 6oi-hydroxymethyl3,8,5oz,6,6 trihydroxypreguan-Z(l-one when tested in the followingassay:

scribed by M. L. Anson in J. Gen. Physiol, 22, 79 (1938) and isdependent upon the fact that the proteolysis of p The technique employedis a modification of that del hemoglobin results in liberation ofpeptides containing 40 tyrosine and tryptophane units characterized byan ab- 0H CHzOR sorption band in the ultraviolet spectrum at 275 milli-H microns. Absorption at this wave length serves there- Wherein X can bea carbonyl, B hydrOXymethy1ene or fore as an index of the extent towhich the proteolysis hydrocarbonoxymethylena group in which the hydrohas occurred. Each test is carried out in 4 test tubes carbon moiety isreadily removed, benzyl, and R to which are added solutions, measured inml., of hemocan be hydrogen or a lower alkanoyl radical. p p p f fhydfochlonq Field P Examples of the lower alkanoyl radical encompassedChlonc f addltlons made p ly by the R term are fo y] acetyl, propionylbutyryl, fore and immediately after simultaneous incubation at valeryl,caproyl, heptanoyl and the branched chain 37 in accordance with theschedule shown in Table I. groups isomeric therewith. TABLE I Thecompounds of this invention are conveniently Additions afterzhomsmanufactured by a process which involves cleavage of Additions atOtimeat 37 the 50:,6m-6POXY ring of the novel intermediates represented bythe following structural formula CH O-GH:

TesttubeNo. Hgb Pep Cpd HCl Opd H01 H0104 The hemoglobin solution (Hgb)is prepared by mixing O OH 60 g. of Hemoglobin Substrate Powder (bovine)[Worthington Biochemical Corporation, Freehold, N.J.] with 2000 ml. ofdouble distilled water, successively filtering and centrifuging theresulting slurry, adjusting the pH of the saturated solution thusseparated to 2.0 with 6 N @omo hydrochloric acid, and finally dilutingwith an equal vol- I ume of pH 2.0 hydrochloric acid, prepared asdescribed 0 below. The pepsin solution (Pep) is prepared by dissolving4.0 mg. of 3X Crystallized Pepsin (hog) [Pentex, In-

wherein R is hydrogen or a lower alkanoyl radical. That corporated,Kankakee, 111.] in 200 ml. of pH 2.0 hydrocleavage is effected byreaction with a suitable acidic chloric acid and diluting 6.25 ml. ofthe resulting solureagent such as perchloric acid, acetic acid, periodicacid, tion with a further quantity of pH 2.0 hydrochloric acid sulfuricacid, boron trifluon'de or acid-washed alumina. q.s. 25 ml. The compoundsolution (Cpd) is prepared by mixing mg. of compound with 5.0 ml. of pH2.0 hydrochloric acid and filtering out any material which remainsinsoluble. The hydrochloric acid s lution (HCl) is prepared by dilutingconcentrated hydrochloric acid to pH 2.0 with double distilled water.The perchloric acid is prepared by diluting concentrated perchloric acidwith double distilled water q.s. 20% by volume. When the additions afterincubation have been completed, the contents of each tube is filtered toremove undigested protein precipitatedby the perchloric acid (which alsoserves to inactivate the enzyme), 1 ml. of each filtrate is diluted withml. of pH 5 sodium acetate buffer and the resulting solutions aresubjected to U.V. spectrophotometric analyses whereby. the absorptionsat 275 millirnicrons are determined. Among the four values thus obtainedfor each compound tested those deriving the tube numbers 1 and 2 serveas controls, being representative of absorption due to peptides producedby incubation of uninhibited enzyme and substrates superimposed uponabsorption due to compound itself, whereas those deriving from tubenumbers 3 and 4-so called treats-represent absorption due to peptidesproduced by incubation of enzyme and substrate in the presence ofcompound, superimposed upon absorption due to compound itself. Acompound is considered pepsindnhibiting if the mean treat value (treat)is significantly (P 0.05, Students t-test) less than the mean controlvalue (control) therefore. Pepsin is known to play a causal role in theproduction of peptic ulcer.

Illustrative of the anti-protozoal property of these compounds is theactivity of 6ot-acctoxymethyl-3,8benzyloxy- 5a,6;8-dihydroxypregnan 2Oone and 6cx-3CE1ZOXYH16thYl- 3,8,5a,6fi trihydroxypregnan-ZO-one whentested in the following assay:

A sterile nutrient medium composed of 12 g. of proteose peptone, 8 g. ofsucrose and 1000 ml. of distilled water is inoculated with a viableaxenic culture of T errahymena gelleii, then is incubated at roomtemperature for 24 hours. At the end of that time a 0.5 ml. quantity istransferred aseptically to a test tube containing approximately 5 mg. ofthe test compound. A test tube c ntaining the culture alone serves as acontrol. The tubes are incubated at room temperature for 24 hours, thenare examined microscopically in order to determine the degree of growthof the microorganisms. A compound is considered active if it results ina definite inhibition of growth as compared to the control.

The invention will appear more fully from the examples which follow.These examples are given by way of illustration only and are not to beconstrued as limiting the invention either in spirit or in scope as manymodifications both in materials and methods will be apparent to thoseskilled in the art. Temperatures are given in degrees centigrade C.) andquantities of materials in parts by weight except where otherwise noted.

EXAMPLE 1 A 50% sodium hydride in mineral oil suspension in the amountof 7.2 parts is washed several times with hexane in order to remove theoil and the sodium hydride thus obtained is dried, then mixed thoroughlywith 32.8 parts of trirnethylsulfoxonium iodide. The temperature of thatmixture is then lowered to approximately 15 and 170 parts by volume ofdimethylsulfoxide is added, cautiously at first, with stirring. Aftercompletion of the addition, the mixture is warmed to room temperatureand stirred for about 30 minutes. The temperature is again lowered toapproximately 15 and 35 parts of3fi-benzyloxy-5othydroxypregnane-6,20-dione ZO-ethylene ketal is added.The temperature of the reaction mixture is allowed to rise toapproximately 25, then is stirred for approximately 24 hours. Dilutionof that mixture with approximately 1000 parts of water results inprecipitation of the solid product, which is collected by filtration,then extracted into methylene chloride. The organic solution thusobtained is filtered through a mixture of sodium sulfate anddiatomaceous earth, then is diluted with hot hexane and partiallyconcentrated. Cooling of the mixture results in crystallization of thepure product, which is collected by filtration, washed on the filterwith hexane and dried to alford 3fl-benzyloxy5e,6nt-epoxy 6dhydroxymethylpregnan-ZO-one 20-ethylene ketal, melting at about 150- 152and displaying an infrared absorption maximum at about 2.9 microns andalso nuclear magnetic resonance peaks at about 42.5 62.5, 76, 111, 216,234, 272 and 438 cycles per second. It is represented by the followingstructural formula:

CH3 O-GHz t EXAMPLE 2 To a mixture of 30 parts of pyridine and 30 partsof acetic 'anhydride is added 15 parts of 3I3-bE-HZYIOXY-50t, 6ot-epoxy6p hydroxymethylpregnan-20-one ZO-ethylene ketal and the resultingreaction mixture is stored at room temperature for about 24 hours.Dilution of the mixture with Water results in precipitation of the solidproduct, which is isolated by filtration, then washed with water on thefilter and dried. The resulting 6,8-acetoxymethyl-3fibenzyloxy 5a,6ot-epoxypregnan-20-one 20-ethylene ketal exhibits an infrared absoptionmaximum at about 5.72 microns and nuclear magnetic resonance peaks atabout 42.5, 62.5, 76, 123, 233, 243, 272 and 438 cycles per second. Itis represented by the following structural formula:

I 5" cr-nocoon;

EXAMPLE 3 When an equivalent quantity of propionic anhydride issubstituted in the procedure of Example 2, there is pr0- duced 3 {3benzyloxy 511,6m-epoxy-6,8-propionoxymethyl pregnan-20-one ZO-ethyleneketal.

EXAMPLE 4 To a solution of 10 parts of6,3-acetoxymethyl-3fl-benzyloxy-5a,6a-epoXypregnan-20-0ne 20-ethyleneketal in 80 parts of acetone is added, with stirring, 2 parts by volumeof aqueous perchloric acid dissolved in 10 parts of water. The reactionmixture is stirred at room temperature for about 30 minutes, at the endof which time an additional 10 parts of Water is added. After stirringfor an additional 3 hours, a further 10 part quantity of water is addedand stirring is continued for 21 hours longer. At the end of that timethe mixture is concentrated under reduced pressure to remove themajority of the organic solvent and approximately 200 parts of water isadded. The resulting precipitated solid is collected by filtration,washed on the filter with water and dried to afiord 6 acetoxymethyl-Sd-benzyloxy-5,6-dihydroxypregnan-ZO-one ZO-ethylene ketal.Recrystallization of that epimeric mixture from chloroform-hexaneaffords 6ot-ace toxymethyl-35-benzyloxy-5a,6B-dihydroxypregnan-20-one,melting at about 171-173 and displaying infrared absorption peaks atabout 2.88, 5.8 and 5.9 microns and also nuclear magnetic resonancepeaks at about 38, 73, 126, 250, 254, 282.5 and 438 cycles per second.This compound is represented by the following structural formula:

Further recrystallization from chloroform-hexane of the materialobtained from the aforementioned chloroform-hexane filtrate results in6fi-acetoxymethyl-3fi-benzyloxy-5a,6a-dihydroXypregnan-20-one.

EXAMPLE 5 When an equivalent quantity of 3B-benzyloxy-5a,6aepoxy 6,3propionoxymethylpregnan--one 20-ethylene ketal is substituted in theprocedure of Example 4, there are produced 3 3 benzyloxy-5a,68-dihydroxy-6a-propionoxymethylpregnan-ZO-one and3fi-benzyloxy-5a,6a-dihydroxy-6B-propionoxymethylpregnan-20-one.

EXAMPLE 6 A mixture containing 20 parts of 6a-acetoxymethyl-3B-benzyloxy-5a,6B-dihydroxypregnan-20-one, 2 parts of 5%palladium-on-carbon catalyst and 250 parts by volume of 5% aqueousacetic acid is shaken in an atmosphere of hydrogen at atmosphericpressure and room temperature until one molecular equivalent of hydrogenis absorbed. At the end of that reaction period the catalyst is removedby filtration and the filtrate is concentrated to an oil under reducedpressure. Crystallization of that oil from ether affords a solidproduct, which is further purified by recrystallization fromchloroform-ether-ethyl acetate, thus producing6a-acetoxymethyl-3B,5u,6;3-trihydr0xypregnan- 20-one, melting at about193195. This compound exhibits infrared absorption maxima at about 2.85,2.95 and 5.71 microns and also nuclear magnetic resonance peaks at about48, 73, 127, 142, 250 and 255 cycles per second. It is represented bythe following structural formula CH3 b=0 CH; IW

l ----CH2OCOCH; H on EXAMPLE 7 The substitution of an equivalentquantity of 3fi-benzyloxy 511,619 dihydroxy-6a-propionoxymethylpregnan-20-one in the procedure of Example 6 results in 313,5u,6fl-

trihydroxy-6a-propionoxymethylpregnan-20-one.

EXAMPLE 8 To a solution of 5 parts of 6a-acetoxymethyl-3/3,50:,65-trihydroxypregnan-ZO-one in 80 parts of acetone is added,

at about 10, 5 parts by volume of an aqueous solution, 8 N in chromiumtrioxide and 8 N in sulfuric acid. The resulting reaction mixture isstirred for about 8 minutes, at the end of which time the excess reagentis destroyed by the addition of 8 parts of isopropyl alcohol. Dilutionof the mixture with water followed by distillation of the organicsolvent under reduced pressure results in precipitation of the solidproduct. That product is collected by filtration, Washed with water onthe filter and purified by recrystallization from aqueous acetone toyield 6u-acetoxymethyl 501,613 dihydroxypregnane 3,20-dione. Thiscompound melts at about 222-224 and exhibits infrared absorption peaksat about 3.8, 3.92, 5.7, 5.76 and 5.81 microns and also nuclear magneticresonance peaks at about 41, 84, 137 and 250 cycles per second. It isrepresented by the following structural formula:

OH CHzOCOCH;

EXAMPLE 9 By substituting an equivalent quantity of 3B,5u,63-t1ihydroxy-6ot-propionoxymethylpregnan-ZO-one and otherwise proceedingaccording to the processes described in Example 8, there is produced5u,6fl-dihydroxy-6a-propionoxymethylpregnane-3,20-dione.

EXAMPLE 10 To a solution of 3.1 parts of 6a acetoxymethyl-35,5a,6B-trihydroxypregnan-20-one in parts of methanol is added 3 partsby volume of 15% isopropanolic hydrogen chloride and the resultingreaction mixture is allowed to stand at room temperature for about 16hours. At the end of that time the organic solvent is removed bydistillation under reduced pressure and the residual mixture is dilutedwith water. The semi-solid product thus obtained is collected byfiltration, then dried and recrystallized from chloroformethyl acetateto yield 36,51,65- trihydroxy-6ot-hydroxymethylpregnan 20 one,characterized by infrared absorption maxima at about 2.91 and 5.88microns and also nuclear magnetic resonance peaks at about 38, 88,122.5, 225, 235, 253 and 263 cycles per second. This compound isrepresented by the following structural formula:

7 What is claimed is:

1. A compound of the formula:

---CH:OR OH V ---CH2OR' wherein Z is selected from the group consistingof carbonyl and fl-hydroxymethylene radicals and R is a lower alkanoylradical.

3. As in claim 1, a compound of the formula:

---OHaOR wherein R is a member of the class consisting of hydrogen andlower alkanoyl radicals.

4. A compound according to claim 1, wherein X is 8- benzyloxymethyleneand R is acetyl, that compound being 6a-acetoxymethyl-BB-benzyloxySa,6fi dihydroxypregnan-ZO-one.

5. A compound according to claim 1, wherein X is 18- hydroxymethyleneand R is acetyl, that compound being 6u-acetoxymethyl-3{3,5a,63-trihydroxypregnan-ZO-one.

6. A compound according to claim 1, wherein X is carbonyl and R isacetyl, that compound being6a-acetoxymethyl-5a,6fi-dihydroxypregnane-3,ZO-dione.

7. A compound according to claim 1, wherein X is ,8- hydroxymethyleneand R is hydrogen, that compound be ing 6a-hydroxymethyl 3,6,5a,6 8trihydroxypregnan-ZO- one.

8. A compound of the formula:

on. o-om o-crn 6" onion wherein R is selected from the group consistingof hydrogen and lower alkanoyl radicals.

9. A compound according to claim 8, wherein R is hydrogen, that compoundbeing 3,8 benzyloxy-5a,6aepoxy-Gfl-hydroxymethylpregnan 20 one20-ethylene ketal.

10. A compound according to claim 8, wherein R is acetyl, that compoundbeing 6fi-acetoxymethyl-3,H-benzylcity-5a,Ga-epOXypregnan-ZO-OneZO-ethylenc ketal.

References Cited Graber et al., Journ. Org. Chem, vol. 27, July 1962 pp.2534-41.

LEWIS GO'ITS, Primary Examiner E. G. LOVE, Assistant Examiner US. Cl.X.R.

Patent No. Dated AD ril 28. 1970 Inventor(s) Leonard N. Nysted It iscertified that error and that said Letters Patent are Column 7, firstformula appears in the above-identified patent hereby corrected as shownbelow:

W should be H II Column 7, second formula should be SIGNED AND m! m AUG1451970 (SEAL) Attest: "Ill-IAN E- m, JR.

Gomiaaiom or M L Edward M. Fletcher. 1:.

A testing Offiour

